PROJECT SUMMARY/ABSTRACT B-1 cells are murine innate immune B cells that preferentially localize in the peritoneal cavity, secrete natural antibodies in a T cell independent manner, and play important roles in the first line of defense against bacterial or viral infections, sepsis, and atherosclerosis. Despite the importance of B-1 cells in host defense and homeostasis, their developmental origin is not clear. CD5+B-1a cells residing in the peritoneal cavity can be reconstituted by transplantation of fetal liver (FL) progenitors, but not by adult bone marrow (BM) hematopoietic stem cells (HSCs). Thus B-1a cells are considered to be of fetal origin and the mechanisms that maintain B-1a cells in adult life without being replenished by adult HSCs have yet to be elucidated. The overall goal of the project is to determine the contribution of HSC-independent embryonic B-1 progenitor cells into the adult peritoneal B-1a cells and define the mechanisms that maintain B-1a cells throughout adult life. We have reported the presence of B-1 progenitor cells originated from hemogenic endothelial cells (HECs) in the yolk sac (YS) in an HSC-deficient mouse model. Our recent preliminary data demonstrated that fetal liver HSCs poorly reconstituted B-1a cells upon transplantation. Thus, we hypothesize that peritoneal B- 1a cells are derived from HECs and are totally independent of HSCs contributions at any stage of development or adult life. In Aim 1, we will utilize a lineage tracing mouse model where one can mark HECs and their progeny at a specific embryonic time point before HSC emergence by tamoxifen induction and we will supplement this approach with HSC transplantation assays from fetus to neonatal and adult stages to prove there is no contribution of HSCs into the B-1a cell pool. In Aim2, we will interrogate the molecular mechanisms that maintain the self-replenishing ability of embryonic derived B-1a cells throughout life. Bmi1 is a polycomb group protein that is critical for self-renewal of HSCs. We now report that the self-replenishing capacity of Bmi1-/- and CD19Cre:Bmi1flox/flox B-1a cells is impaired, raising the hypothesis that Bmi1 plays an important role in B-1a cell self-renewal. Since Bmi1-/- mice displayed a significantly more severe reduction in B-1a cell number compared to CD19Cre:Bmi1flox/flox mice, we hypothesize that the B-1a cell microenvironment may be altered in Bmi1-/- mice. We will examine a niche for B-1 cells, the recently reported fat-associated lymphoid clusters (FALCs) in the omentum and mesentery, in Bmi1-/- mice. We will also pursue a downstream target of Bmi1 as playing a role in B-1a self-renewal. These experiments consider cell intrinsic and extrinsic roles of Bmi1 to define its critical role in B-1a cell maintenance and self-renewal. Our results will establish a new paradigm for understanding B-1 cell development and maintenance in mice and will provide new tools to translate to human investigation of B-1 like cells. 1